Culture techniques to grow anti-tumor CD8 T cells for adoptive therapy are available for mouse tumor models and human melanoma patients; in contrast, reliable culture techniques to grow anti-tumor CD4 T cells with therapeutic characteristics are not yet available. Mouse data indicate that anti-tumor CD4+ T cells of "Th1-type" (interleukin-2 and interferon- gamma secreting) may potentiate the therapeutic effects currently possible with anti-tumor CD8+ T cells; hence, a major effort to develop reproducible CD4+ culture techniques is underway. In mouse work we have shown that dendritic cells (DC) pulsed with tumor lysates are potent activators of tumor-specific CD4+ T cells in culture. CD4+ T cell lines have been established which specifically recognize tumor, have "Th1" characteristics, and have therapeutic effects when given to tumor-bearing mice. Improved culture reproducibility is being achieved by removing foreign species proteins from culture and by improving DC performance through differential upregulation of costimulatory molecules B7-1 and B7-2. We are optimizing adoptive therapy with CD4+ T cells to maximize tumor rejection with or without coadministration of CD8+ T cells. Established mouse anti-tumor CD4+ T cells also provides a source of T cell receptor for molecule studies; Dr. M. Nishimura has utilized anti-sarcoma CD4+ T cells as T cell hybridoma fusion partners to aid in tumor antigen isolation. In parallel work we have attempted to grow antigen-specific human CD4 T cells from melanoma and colon cancer patients as well as from normal donors. We have perfected elutration techniques in collaboration with Charles Carter (DTM) which permit the enrichment of DC from human blood; these DC can be utilized to process and present both recall antigens and-tumor antigen to autologous CD4+ T cells. CD4 responses to recall antigens are readily demonstrable in normal donors and melanoma patients, but impaired in colon cancer patients. In addition, it has been difficult to demonstrate an anti-tumor response. We have identified several reasons for these difficulties: (1) cultured human DC require cytokine activation in culture to increase 57 and MHC Class II express (2) colon cancer patients appear to have an additional impairment at the. level of CD4+ function lacking in melanoma patients. We are.addressing and correcting these dysfunctional components to generate tumor-specific Th1- type CD4 T cells in cancer patients.